The antibiotic 11837 r.p. and process for its manufacture using streptomyces viridans

ABSTRACT

The invention provides the new antibiotic 11837 R.P., which is useful in treating infections caused by Gram-positive microorganisms, and its production by fermentation using the newly discovered microorganism Streptomyces viridans(NRRL) 3087).

United States Patent Mancy et al.

[ 51 Aug. 22, 1972 [54] THE ANTIBIOTIC 11837 R.P. AND

PROCESS FOR ITS MANUFACTURE USING STREPTOMYCES VIRIDANS [72] Inventors: Denise Mancy, Charenton; Leon Ninet; Jean Preud'Homme, both of Paris, all of France [73] Assignee: Rhone-Poulenc S.A., Paris, France [22] Filed: Sept. 16, 1964 [21] Appl. No.: 399,129

[30] Foreign Application Priority Data Sept. 19, I963 France ..63948030 Nov. 22, 1963 France ..63954708 [52] US. Cl ..424/ll8 [51] Int. Cl. ..A6lk 21/00 Primary Examiner-Albert T. Meyers Assistant Examiner-Daren M. Stephens Attorney-Stevens, Davis, Miller & Mosher [57] ABSTRACT The invention provides the new antibiotic l 1837 RR, which is useful in treating infections caused by Grampositive microorganisms, and its production by fermentation using the newly discovered microorganism Streptomyces viridans(NRRL) 3087).

18 Claim, 2 Drawing Figures SHEET 1 [If 2 PATENTEU M1822 I972 on 8 E. ow 2. 02

Denise. Mane; Lean "ma/i jean preud Ham 1e,

Inventor 1-004 M"M Allorm'ys QVM PATENTEDwszz m2 SHEET 2 0F 2 Dam sa. Mane,

Leon N/neni' Jean Pram-l Ham/ms Inventor Attorneys THE ANTIBIOTIC 1 1837 R.P. AND PROCESS FOR ITS MANUFACTURE USING STREPTOMYCES VIRIDANS THIS INVENTION relates to antibiotics and their manufacture.

The invention provides a new antibiotic, hereinafter referred to as 11,837 R.P., and a process for its manufacture. This new antibiotic is of special value because of its considerable antibacterial activity towards Grampositive microorganisms and the very long period for which it remains active. It is obtained from culture media of the microorganism which is characterized in detail below, belonging to the streptomyces genus and designated as Streptomyces viridans DS 9466 (NRRL 3087).

11,837 R.P. is very readily soluble in water, soluble in methanol, pyridine, acetic acid and dimethylformamide, and sparingly soluble or insoluble in ethanol, acetone, chloroform and n-hexane. An aqueous solution of 11,837 R.P. is very stable at pH 5 to (keeping at least 90 percent of its activity for a fortnight at 37C.), moderately stable at pH 4 (losing 30 percent of its activity within a fortnight at 37C.) and relatively unstable at pH 2 (losing 70 percent of its activity within 6 days at 37C.

11,837 R.P. gives negative test results in the following reactions: biuret, ferric chloride, and ninhydrin; and positive test results in the following reactions: ninhydrin after acid hydrolysis, diamtization, xanthoprotein, ammoniacal silver nitrate solution (doubtful before hydrolysis, very strong after acid hydrolysis), phloroglucinol, carbamle, potassium permanganate, and with Benedict's reagent after acid hydrolysis. 1t tums concentrated sulphuric acid orange and concentrated hydrochloric acid pale pink.

11,837 R.P. is a strong acid whose neutralization equivalent, measured by potentiometn'c titration with sodium hydroxide, is 600 (pKa 4.1). Its molecular weight appears to be greater than 5,000 because it does not dialyze through a diaphragm of regenerated cellulose (of the Cellophane" type).

11,837 R.P. contains carbon, hydrogen, oxygen, nitrogen and phosphorus. Its elementary composition (as revealed by the analysis of its sodium salt) is approximately: C 46.9%, H 7.9%, O 39.2%, N 3.9%, and P 2.25%. It has the following physical properties. It is a white, amorphous powder, of no definite melting point, but decomposing above 160C. lts infrared spectrum (measured on a tablet of a mixture with KBr) is shown in FIG. 1 of the accompanying drawings, where the upper scale on the abscissa is the wavelength in microns and the lower scale the wave number in cm.', and the transmission in 9b is shown on the ordinate. The principal infra-red absorption bands of 1 1,837 R.P. are as follows:

3360 vs 1 164 m 2930 s 1125 sh 1720s 110-0m where 1630 vs 1068 vs vs very strong 1564 In 1040 vs s= strong 1550 r 968 m rn m medium 1430 sh 950 m w weal:

1400 sh 890 w sh shoulder. 1380 s 860 w The salts, especially the alkali metal salts, of 11,837 R.P. with bases are within the scope of the invention. The sodium salt of 11,837 R.P. has the elementary composition: 0 45.0%, H 7.4%, 0 37.6%, N 3.72%, P 2.16% and Na =4.15%. It has the following physical properties. It is an almost white, amorphous powder, having an optical rotation of {ab} =+6:t1(c 0.6, water). Its ultraviolet spectrum (measured in a solution of 30 mg. per liter of water; i.e., 0.003 percent) shows end absorption: 220 mp. (E,'",,,, 30) and 257 mp. (E 3.5). Its infra-red spectrum (measured on a tablet of a mixture with KBr) is shown in FIG. 2, where the lower scale on the abscissa is the wavelength in microns and the upper scale shows the wave numbers in 6111.", and the transmission in percent is shown on the ordinate. The principal infrared absorption bands of this salt are as follows:

3400 vs 1 160 m 2910 s l m 1730 vs 1062 vs where 1547 m 1042 sh vs very strong 1525 s 1030 sh s strong about 1430 sh 972 m m medium 1378 s 947 m w=weak 1330 m 890 w sh shoulder The bacteriostatic activity of 11,837 R.P. towards a number of microorganisms has been determined by a conventional dilution method. For every microorganism the minimum concentration of antibiotic was measured at which under defined conditions all visible development of the microorganism on a suitable nutrient broth was inhibited. The results of the various measurements are summarized in Table l where the minimum bacteriostatic concentrations of the new antibiotic are shown in micrograms of substance per cc. of test nutrient medium.

These different measurements show that the bacteriostatic activity of 1 1,837 R.P. is directed principally towards microorganisms which accept Gram staining, its activity against Streptococcus hemolytr'cus being particularly strong. It is relatively inactive against Gramnegative microorganisms though its activity against Neisseria catarrhalis, Neisseria gonorrhaeae and Bruce!- la abortus' bovis is appreciable. No cross-resistance is noticeable with the following antibiotics: penicillin, streptomycin, tetracycline, chloramphenicol, spiramycin, carbomycin, erythromycin, pristinamycin and novobiocin. The results of various measurements of bacteriostatic concentrations of 11,837 R.P. against different strains of Staphylococcus that are resistant to one or several of the above-mentioned antibiotics, are summarized in Table II where there are shown for purposes of comparison the bacteriostatic concentrations for three strains of Staphylococcus sensitive towards all these antibiotics (also listed in Table 1).

Sarcr'na lutea ATCC 9341 Streptococcus faecalis ATCC 9790 Streptococcus vindanr Institut Pasteur) Streptococcus p yogener hemolyticur (strain Dig 7, lnstitut Pasteur) Neisreria gonorrhaene (A 50 lnstitut Pasteur) Diplococcus pneumonia:

(strain Til, lnstitut Pasteur) 0.03 Bacillus rubtilir ATCC 6633 3 TABLE [1 Minimum bacterioststic concentration in .lec. dfls Strain of Staphylococcus oureur tested Strain 209 P-ATCC 6538 P (s) Strain 209 P. rendered resistant to spiramycin 0.2 Strain 209 P, rendered resistant to carbomycin 0.15 Strain 209 P, rendered resistant to pristinarnycin 0.25 Strain 209 P, rendered resistant to novobiocin 0.1 Strain 133 (lnstitut Pasteur) (s) 0.15 Strain Smith (I) 0.35 Strain B, (resistant to penicillin and streptomycin) 0.2 Strain Hb (resistant to penicillin and tetracycline) 0.3 Strain Beaujon 3 (resistant to penicillin ltreptomycine, tetracycline and chloramphenicol) 0.3 Strain MB 1 (resistant to penicillin and erythromycin) 0.1 Strain Lavault (resistant to penicillin, erythromycin and spiramycin) 0.1

The antibacterial activity of 11,837 R.P. has been shown in in vivo tests on laboratory animals infected with microorganisms such as streptococci, Pneumococci, Staphylococci and Neisseriae (Neisreria meningitidzlr). It is particularly potent in the mouse on parenteral administration, the activity by the intravenous route being twice that of administration by the subcutaneous route.

It has a very prolonged activity which makes it a very good prophylactic agent. This prophylactic activity has been demonstrated towards staphylococcal and streptococcal infections of the mouse. Thus, for example, a single dose of 250 mgjkg. of 11,837 R.P. given intravenously protects all mice from intraperitoneal infection by streptococci for 56 days after administration of the antibiotic. The same dose (250 mg/kg.) introduced by the subcutaneous route of benzathine penicillin G protects the mouse only for 24 hours.

The toxicity of 11,837 R.P. has been studied in the mouse. The 50 percent lethal dose (LD has been 6S determined both for subcutaneous and intravenous administration:

LB 1.7 g./kg. (subcutaneous) 1.5 gjkg. (intravenous). As these values show, the antibiotic is of very low toxicity.

The microorganism that produces the antibiotic 11,837 R.P. belongs to the genus Streptomyces and is called Streptomyces viridans DS 9466. It has been deposited at the laboratory of the NRRL at Peoria, 111., United States of America under reference number NRRL 3087.

This organism was isolated from a soil specimen, collected near Madras, India, in the following manner. The soil specimen was suspended in sterile distilled water and this suspension was then diluted to different concentrations. A small volume of each dilution was placed in Petri dishes each containing an agar nutrient medium. After several days incubation at 26C. the colonies of microorganisms to be isolated were pricked out onto agar slopes to obtain more abundant cultures.

According to the classification in Bergeys Manual of Determinative Bacteriology (7th Edition [1957} The Williams & Wilkins Company, Baltimore) for the genus Streptomyees and the classification and descriptions given by S. A. Waksman in The Actinomycetes (The Williams & Wilkins Company, Baltimore [1961]) it is found that the morphological characteristics of the organism that produces 11,837 RP. correspond to those described for Streptomyces viridans. Therefore the organism which produces 11,837 RP. has been grouped with this species and called Streptomyces viridans DS 9466.

Table 111 lists the characteristics of the strain Streptomyces viridans DS 9466 side by side with the description of the species Streptomyces vin'dans in Bergeys Manual of Determinative Bacteriology. This comparison shows clearly that the organism which produces 11,837 R.P. displays characteristics corresponding to those described for Streptomyces viridans. The minor difl'erences shown can be attributed only to difl'erent strains of the same species.

Streptomyces viridam DS 9466 is capable of producing on certain culture media, particularly Bennetts medium, a soluble green pigment which, at the start of this product and when a sufficient quantity thereof has been produced, displays an intense emerald green color. This product of an emerald green pigment is rather capricious and depends mainly on the intensity of the seeding of the microorganism on the medium where it is produced; the presence of soil favors it; the emerald green color of the substrate does not persist and, as the culture ages, its color changes according to the culture medium and the quantity in which it is produced, to blackish green or brownish green or greyish brown.

TABLE 1H Streptomycer Bergey's Manual of Deterl'nlnltive Bacteriology, Streptornyees viridaru 7th Edition DS 9466 pages 758-759 Vegeta- Colonies Vegetative mycelium greyish yellow live green to to vivid yellow or brownish yellow. growth greenish On certain media it takes a green brown color turning togreenish brown or blackish green in the zone: where soluble green pigment is produced.

Aerial Deep grey Light grey to dark grey. powdery,

myceliuln tinged with well developed bong lporophorea,

olive or monopodial, ending in spore chains green-grey, inserted in terminal bunches; velvety, susceptible of branching along covering the their main stem. Spore chains whole colony. llflexuous or forming short spirals. Long aporo- Cylindrical spores. phorea forming spirals.

Cylindrical spores.

Gelatin Rapid Moderate liquefaction.

Agar liquefaction. Vegetative growth yellow or browngrowth ish to green-brown, according to brown-green medium used. Soluble pigment rlil soluble or brownish; in addition in brown pigment certain cases facultative and is produced more or leg regular production of a soluble green pigment.

Syn- Green Vegetative myceliurn yellowish or thetic colonies. brownish, turning green when agar Production soluble green pigment I produced.

of soluble Soluble pigment nil or yellowish green pigment. to brownish, mostly of low a. intensity. ln certain cases limited production of soluble green pigment (glycerin aaparagine agar, Waksman'a starch agar) or more abundantly (Cupek's glycerin agar; in the latter case, the presence ofsoil has proved favorable.

Milk Coagulated Slow pcptoniaation, without prior and quickly coagulation at 25, with slight peptonized. coagulation at 37'.

Sucrose Rapid Poor utilisation.

inversion Starch Rapid Hydrolysis fairly rapid.

hydrolysis.

Cellu- Poor growth. Positive growth.

lose

Ni- Active Rapid and massive production tritea production from nitrates.

from nitrates nistic Nil Production of I 1,837 R.P.

ties

Streptomyces viridam DS 9466 displays a sporulated aerial system of rather dark grey color, which develops well on a number of conventional culture media, especially on Bennett's medium. It forms cylindrical spores of 0.7 to 0.9,. width and 1.3 m 1.5 length. These wise indicated, they refer to cultures grown for two to four weeks at 26C., when a good state of development had been reached. Most of the culture media employed were prepared as directed in "The Actinomycetes", S. A. Waksman, pages 193-197, Chronica Botanica Company, Waltham, Mass, United States of America, 1950; where this applies, there will be found the letter W followed by the number accorded to them in The Actinomycetes.

The references or compositions of the other culture media are as follows:

Ref. A K. L. Jones Journal of Bacteriology 57, p.

Ref. B A. M. Williams and E. McCoy Applied Microbiology I, p. 307 [1953].

Ref. C Grundy et al. Antibiotics and Chem. 2, p.

Ref. D --O.5 percent peptone +013 percent meat extract +0.5 percent tyrosine ----2 percent agar.

Ref. E Inorganic salts starch agar T. G. Pridham et al, Antibiotics Annual, 1956-1957, pp. 947-953.

Ref. F corresponding to formula W-l except that 30 g. of sucrose are replaced by 15 g. of glycerin.

Ref. G Plain gelatin, prepared as directed in Manual of Methods for Pure Culture Study of Bacteria" of the Society of American Bacteriologists, Geneva, N.Y., 11 18.

Ref. H Manual of Methods of Pure Culture Study of Bacteria Society of American Bacteriologists, Geneva, N.Y., 11 -18.

Ref. 1 Manual of Methods of Pure Culture Study of Bacteria Society of American Bacteriologists, Geneva, N.Y., 11 19.

Ref. J corresponds to formula W-l 8, except that the sucrose is omitted and replaced by small strips of filter paper dipped partially in the liquid.

Ref. K Commercial skimmed milk powder, reconstituted as instructed by the manufacturers.

Ref. L H. D. Tresner and F. Danga Journal of Bacteriology 76, pp. 239-244l1958].

TABLE IV Degree of Vegetative mycelium or Aerial system (including Remarks and biochemical Culture medium developunderside of culture total of aerial mycclium Soluble pigment properties ment and sporulationj Bennett's agar (Ref. A)... Good. Lnderside greyish yellow, Light grryish to dark Green, when ageing Production of soluble turning to greenish grey. Well developed. turning greenish grey green pigment is pr0- brown ln spots where to very dark grr-enmeted when soil present. soluble green pigment brown, and blackish. is formed. depending on quantity devolopnd. M lw yp one agar ...do..-. Underslde g'reyish yellow Whltlsh to grey Slight hrownlsh grey (Ref. B). brown,

Emerson's agar (l'i'-23) Medium. Light yellowish brown. whitish, in. traces. Nil

Well developed.

Glucose-[leptons agar ..do Light-yellow. Rather whitish. Very moderate. Slight yvllowish well developed. Utl'l t agar (f-5) Poor Light g-reyish yellow. Nil Nil Moderate development.

TABLE IV Degree of Vegetative mycelium or Aerial system (including Remarks and biochemical Culture medium developunderside of culture total of aerial mycelium Soluble pigment properties merit and sporuiation) After tortniht rTl. m'wfluk yellow.

lumafiarfiehg Medium Vivid yellow to brownish After fortnight nil.

(W-2). v After 1 month small After 1 month small (whitish to grey'ish) quantity, greyish to in older cultures. greenish grey. Glycerlne-asparagine agar Moderate Brownish yellow with Greyish white to light Slight brownish grey -3) irregular trace of greyish. Very modwith irregular small greenish. crate development. quantity of greenish to greenish grey. Krainskys calcium Good. Underside light greyish Whltish to light grey Nil, or slowly formed, Cfl e u 11151 1 111518! 8 yellow. slight greyish. solubilized. y osine aga (Ref. D) -do. Underside yellowish whitish. Moderately Yellow-brown Tyrosine well solubllised.

brown. developed. Starch agar (W-ll) Moderate. Underside greenish grey whitish to grey. Mod- Greyish green to greenish to dark greenish brown. erately developed. grey. Pridham's starch agar Good L'nderside light grayish Light grey, Fairly well Nil M diu ydr l of (Ref. yellow. developed. starch- Czap k's synth tic ..do Underside light brownish w i h to grey Light brownish yellow The presence of soil has glycerine agar (Ref. F). yellow to yellowish brown. Turning to dark greenish brown at the site where soluble green pigment is produced.

Potato culture (W27). Fairly is" good yellow brown to very dark green, turning blackish brown on ageing.

Medium. Surface culture limited to the site of the point of inoculation. Under- Puenplatin 12% (Ref.

Veget. mycelium greenish whitish to light grey.

Moderate development.

whitish to greyish.

Moderate development.

considerable influence on this medium insofar as production of soluble green pigment is concerned.

to light yellowish brown. When soil is added soluble green pigment is produced which on ageing turns grayish brown to very ark greenish brown, almost blackish. Production is more or less delayed. Abundant. Very dark green, turning greenish black, diflusl into the whole the potato. Delayed production. Alter fortnight nil. After 1 month light Liquefaction of gelatin positive but rather slow, incomplete alter side yellow. yellowish. 1 month- Nitm nutri nt broth Good. whitish ring n surface Very limited (lcvelop- Ni] Strong positiv nitrit (Ref. H). ment. whitish. cfl Dimmicks glucose Moderate" Light ring and veil very Nil or whitish, in traces... Very faint yellow o g positive nitrite broth (Rel. I). moderately developed reaction.

on surface. Greyish white to light yellowish. Czapek's synthetic cellu- Fairly N Grey, well developed in N1] Strong positive ni rit 1058 broth (Rel. J). good. whole of paper proreaction.

truding above protll. Good. Ring well developed. Nil to slight traces, N0 Chang ill appearance Skirnmed milk (Ref. K)

(a) at 26. Very faintly yellowish.

Ring development modcrate. Light yellow to light yellowish brown.

'Iresner and Danga medium (Ref. L)v

whitish.

Nil to faint traces, whitish.

of milk after fortnight. Then slow peptonlsation, almost complete after 1 months cultivation. No coagulation. pll goes from 6.3 to 7.2- 7.4 within 1 month.

.......................... No change in appearance of milk after fortnight. At end of 3 to 4 weeks slight coagulation with beginning peptonisation. pl] goes in 1 month from 6.3 to 5.9-6.1.

. Production of negative.

According to the method of Pridharn and Gottlieb (J. Bacteriol. 56, pp. 107-114 [1948]) Streptomyce: viridam DS 9466 readily utilizes the following as carbon sources: xylose, arabinose, rhamnoee, glucose, galactose, levulose, marmose, lactose, maltose, trchalosc, cellobiosc, dextrin, starch, inulin, erythritol, adonitol, dulcitol and sorbitol; utilization of sucrose is weak and slow, almost nil within days and allows only a poor development within one month.

According to a feature of the invention, the antibiotic herein designated 11,837 R1. is produced by aerobically cultivating Streptomyces vin'dam DS 9466 (NRRL 3087) or a 11,837 R.P.-producing mutant thereof on an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances, and separating the 11,837 R.P. formed during its cultivation.

Srreptomyces viridaru DS, 9466 can be grown by any of the known surface or submerged aerobic culture methods, the latter procedure being preferred because it is more convenient. For this purpose conventional apparatus may be used. in particular, the following sequence of operations may be adopted:

Streplomyces viridans DS 9466- stock culture on agar culture in an agitated flask inoculum culture in fermentation vessels production culture in fermentation vessels.

ble oils such as lard oil or soybean oil may be advantageously used instead of or in admixture with carbon-, hydrogenand oxygen-containing substances.

A very wide range of suitable sources of assimilable nitrogen is available. The source may be very simple chemical compounds such as nitrates, mineral or organic ammonium salts, urea or arninoacids. It may also be in the form of a complex substance containing a proportion of nitrogen, principally in the form of protein, e.g. casein, lactalbumin, and gluten and their hydrolysates, soybean flour, peanut meal, fish meal, meat extracts, yeast extracts, distillers solubles, and comsteep liquor.

Of the inorganic substances added some may have a buffering or neutralizing effect, such as the alkali metal phosphates or alkaline earth metal phosphates, or the carbonates of calcium and magnesium. Others contribute to the ionic equilibrium needed for the development of Streptomyces viridans DS 9466 and the formation of the antibiotic; examples of these are the chlorides and sulphates of the alkali and alkaline earth metals. Finally, some act more especially as activators of the metabolism of Streptomyces viridans DS 9466; to these belong the salts of zinc, cobalt, iron, copper, and manganese.

The pH of the fermentation medium at the start of the culture should be within the range from 6.0 to 7.8, preferably from 6.5 to 7.5. The optimum fermentation temperature is 2528C. but satisfactory production is obtained at temperatures from 23 to 35C. The rate of aeration of the fermentation vessels may vary within rather wide limits, but it has been observed that a particularly advantageous rate is 0.3 to 2 liters of air per liter of medium per minute. The maximum yield of antibiotic is achieved after 4 to 7 days growth, but this period depends predominantly on the individual medium used.

From the foregoing it will be realized that the general conditions of the culture of Streptomyces viridans DS 9466 for the production of the antibiotic 11,837 R.P. may be widely varied and adapted as appropriate to the circumstances.

11,837 R.P. can be isolated from the fermentation media by a variety of methods. in one such method the fermentation medium is filtered at a pH of 7 or more, but under these conditions part of the activity remains in the filter cake and part in the filtrate and both must be treated to extract the active principle. lt is therefore preferable to perform the filtration at a pH BELOW preferably at about pH 3; under these conditions the whole of the activity remains in the filter cake from which it can be extracted at a pH from 3 to 7 by water containing a lower alcohol such as methanol, ethanol or propanol, or with a mixture of lower aliphatic alcohols containing at most six carbon atoms, the most useful mixture containing n-butanol methanol at the ration of 2:1 to 3:1 by volume. It is also possible to percolate the fermentation medium through a column containing a strongly basic anion exchange resin, followed by an elution with an aqueous alcoholic solvent, such as aqueous methanol, containing an electrolyte.

The crude product can be isolated from its alcoholic or aqueous alcoholic solution mentioned above by conccntration of the solution to a small volume, and this concentration is advantageously performed at a temperature below 40C. and under reduced pressure. By

cooling and/or addition of a substance which is a poor solvent for 1 1,837 R.P., such as a ketone, an ether, an ester, a chlorinated solvent, benzene or hexane, the crude antibiotic is caused to precipitate. When the antibiotic is contained in the filtrate from the culture media, the solution is extracted with a water'immiscible solvent such as an aliphatic alcohol containing four or five carbon atoms.

This is followed by concentration to a small volume and precipitation as described above. The antibiotic 11,837 R.P. can then be purified by fixing it on a strongly basic anion exchange resin, followed by elution with an aqueous alcoholic solution containing an electrolyte, such as hydrochloric acid or a chloride of sodium, ammonium, potassium, calcium, or magnesium, added in an amount of 5 to 50 g. per liter of eluant. The eluate can then be concentrated to a small volume at a temperature below 40C. under reduced pressure, the concentrate then being dialyzed against a current of distilled water, using a diaphragm of regenerated cellulose. The mineral salts and other low molecular weight impurities are carried away by the water and 1 1,837 R.P. remains quantitatively in the dialyzed solution. The latter is then subjected to azeotropic concentration to a very small volume under reduced pressure after butanol has been added to it. The purified antibiotic is precipitated from the aqueous concentrate with a mixture of solvents such as acetone isopropanol, if desired with addition of diethyl ether or diisopropyl ether in a ratio such that no separation of phases occurs on mixing with the aqueous phase. A preferred mixture contains acetone and isopropanol in the volumetric ratio of 4:1 or acetone isopropanol ether in the volumetric ratio of 1:1: 1

The antibiotic may then be subjected to a further purification by agitating an aqueous solution of 11,837 R.P. with a strongly acidic cation-exchange resin until a constant pH of from 2 to 3 is reached. The effluent is then washed with a water-immiscible solvent, such as an aliphatic alcohol containing four to five carbon atoms, or an ester such as ethyl acetate, or a mixture of such solvents. The operation is preferably performed with a mixture of equal volumes of n-butanol and ethyl acetate. The aqueous phase is concentrated to one hundredth of its initial volume, whereupon butanol is added and the whole is concentrated under reduced pressure while further butanol is continuously added. Purified 1 1,837 R.P. is obtained by adding a substance that is a poor solvent for 11,837 R.P., such as hexane, centrifuging, and washing and drying the precipitate.

It will be understood that the different methods referred to above can be applied in any desired order or repeated several times as required for the manufacture of 1 1,837 R.P. in a form suitable for the application envisaged.

The following non-limitative Examples illustrate the invention. ln the following the activity is measured biologically by the diffusion method using Bacillus subtilis as the sensitive microorganism and with reference to a sample of pure 11,837 R.P. as standard. This activity is expressed in units (u) per mg. for the solid products and in units per cc. for the solutions (a unit is defined as the minimum amount of 1 1,837 R.P. which, dissolved in 1 cc. of the appropriate medium, inhibits the growth of Staphylococcus aureus 209 1 under specified conditions).

EXAMPLE 1 A l70-liter fermentation vessel is charged with Cornsteep 4.8 kg. glucose hydrate 2.4 kg. sodium chloride 0.6 kg. magnesium sulphate 0.12 kg. water to make I liters.

After the pH value of the mixture has been adjusted to 7.15 by adding 575 cc. of concentrated sodium hydroxide solution (4' 1.33), 0.6 kg. of calcium carbonate are added.

The nutrient medium is then sterilized by bubbling steam at 122C. through it for 40 minutes. After cooling, the broth has a volume of 120 liters and a pH of 7.15. The medium is inoculated with 200 cc. of a culture of the strain Streptomyces viridans DS 9466 grown in an agitated Erlenmeyer flask, and developed for 28 hours at 26C. with agitation and aeration with sterile air, whereupon it is ready for inoculation of the production culture.

The production culture is carried out in a 350-liters fermentation vessel charged with the following substances:

Soybean flour 8 kg. distillers solubles 1 kg. starch 3 kg. soybean oil 3 liters calcium carbonate 2 kg. sodium chloride 2 kg.

cobalt chloride hexahydrate 4 g. water to make l80 liters.

The medium (pl-l 7.05) is sterilized by bubbling steam at 122C. through it for 40 minutes. After cooling, the broth has a volume of 200 liters and its pH is 7.15. Inoculation is performed with 20 liters of the inoculum culture grown in the l70-liter fermentation vessel described above. Culture is performed at 26 to 27C. for 138 hours with agitation and aeration with sterile air, whereupon the pH of the medium is 7.90 and the volume is 180 liters. The amount of antibiotic present in the medium is 4515 u/cc.

EXAMPLE 2 The inoculum culture is grown in a l70-liter fermentation vessel under the conditions described in Example l. The production culture is carried out in a fermentation vessel of 350 liters capacity, charged with the following substances:

Cornsteep 6 kg. starch 2 kg. soybean oil 3 liters cobalt chloride hexahydrate 4 g. water to make 175 liters.

After the pH of the mixture has been adjusted to 5.90 with 380 cc. of concentrated sodium hydroxide solution (d= 1.33), 1 kg. of calcium carbonate is added.

The mixture is sterilized by bubbling steam through it at 122C. for 40 minutes. After cooling, there are finally added to the culture medium under sterile conditions 0.4 kg. of ammonium sulphate in solution in 5 liters of water.

The broth then has a volume of 200 liters and its pH is 6.80. Inoculation is carried out with liters of inoculum culture from the l-liter fermentation vessel. The culture is continued at 26C. to 27C. for 144 hours with agitation and aeration with sterile air. The final pH of the culture is 8.40 and the volume of the fermentation broth 185 liters. The amount of antibiotic then present in the medium is 3550 u/cc.

EXAMPLE 3 Two hundred liters of fermentation medium, containing 2,855 u/oc. at pH 8.4, are adjusted to pH 3 with SN-hydrochloric acid solution in a vat equipped with an agitator, and 12 kg. of a filtration assistant are added. The mixture is filtered through a filter press and the filter cake is washed with 60 liters of tap water. The filtrate and the washings are practically inactive and are discarded. The filter cake is suspended by agitation in liters of a mixture of butanol (2 volumes) and methanol (lvolume). The apparent pH of the mixture is then adjusted to 6 with a ION-solution of sodium hydroxide. Agitation is continued for 30 minutes and the mixture is then filtered through a filter press.

The filtrate is collected and the filter cake washed with 30 liters of the above-mentioned mixture of butanol and methanol. The total of filtrate and washings liters) contains 2075 u/cc. The filter cake is discarded. The alcoholic filtrate is concentrated under reduced pressure (20 mm. Hg) at 35C. to a volume of 2 liters.

The antibiotic which precipitates during the concentration is filtered off, washed with acetone and dried in an oven under a vacuum of 5 mm.Hg. There are thus obtained 254 g. of a greenish grey product containing 1025 u/mg.

EXAMPLE 4 Three hundred and seventy five liters of culture medium at pH 7.9 are strained to remove the coarsest particles. The pH is adjusted to 7 with SN-hydrochloric acid in an agitated vat, and 22.5 liters of Dowex l-X 2resin (Cl'form) are added. Agitation is continued for two hours, and the mixture is then passed through a vibrating sieve. The resin remains on the sieve and the extracted medium is discarded. The resin is then transferred to a column and washed with 50 liters of a 5 percent aqueous solution of sodium chloride and then with 10 liters of water. The washed resin is then dehydrated by passage of 20 liters of methanol, and elution is effected with methanol containing 20 percent water and 2.8 percent of ammonium chloride. The eluates are taken in fractions of 10 liters. The first three eluate fractions contain 98 percent of the eluted activity; they are combined and concentrated to a volume of 3 liters under a pressure of 20 mm.Hg. The concentrated solution contains 92,950 u/cc. and has a specific gravity of 1.057; it is dialyzed for 48 hours against distilled water through a regenerated cellulose diaphragm. After this operation the solution has a volume of 4.7 liters and a specific gravity of 1.025. The solution is concentrated to 300 cc. under a pressure of 5 mm.Hg and then freeze-dried, to yield 48 g. of crude product containing 65 30 u/mg.

EXAMPLE 5 Two hundred and fifty g. of the crude antibiotic prepared as described in Example 3 are dissolved in 4 liters of water at pH 8. The aqueous solution is filtered and then poured through a column of 2.7 liters of Dowex 1-X2 ion exchange resin (Clform), and the effluent is discarded. The resin is washed with 2.5 liters of water and then with 2.5 liters of methanoH-water (80:20 by volume). The antibiotic is finally eluted with 10 liters of methanol+water (80:20 by volume) containing 7.5 g. of potassium chloride per liter.

The eluate is concentrated to 400 cc. under reduced pressure (20mm.Hg) at a temperature below 40C. The concentrate is dialyzed for 24 hours against a current of distilled water through a regenerated cellulose diaphragm to eliminate the mineral salts and low molecular weight organic impurities.

The dialyzed solution (650 cc.) which contains all of the activity of the eluate is concentrated to 125 cc. under a pressure of 20 mm.l-Ig. The resulting concentrate is mixed with 20 volumes of a mixture of ether +acetone+isopropanol (10: 10:10 by volume), and the antibiotic is precipitated. After filtration, washing with acetone and drying overnight at 35C. under 2 mmfig pressure, there are obtained 8.25 g. of a light-brown powder containing 24,600 u/mg.

EXAMPLE 6 7.9 g. of the product of Example are dissolved in 790 cc. of the lower phase separated from a mixture of n-butanol+ethyl acetate-twater (10:10:20 by volume) and agitated to a constant pH of 2.1 with 50 cc. of Amberlite IR 120 ion-exchange resin (acid form) added in small portions. The resin is filtered off and the filtrate washed with 2 X 790 cc. of the upper phase of the above mixture. The washings are then extracted with an equal volume of lower phase.

The lower phases are combined and concentrated to cc. under a pressure of mrn.l-lg. The resulting aqueous concentrate is mixed with 20 volumes of methanol-tether (4:6 by volume). The antibiotic precipitates and yields 3.05 g. of a first crude product containing 18,300 u/mg.

The precipitation mother liquors are concentrated to 100 cc. under a pressure of 20 mm.Hg. There are then added 320 cc. of methanol and 35 cc. of n-butanol. The mixture is concentrated to 15 cc. under 20 mm.l-lg pressure at a temperature below 10C. After adding 15 volumes of n-hexane, centn'fugation, washing with hexane and drying overnight at 35C. under 2 rnml-lg pressure, there are obtained 1.78 g. of purified antibiotic containing 36,000 u/mg.

The combined upper phases are likewise concentrated to about 70 cc. under 20 mm. Hg pressure. By adding 15 volumes of hexane, there are isolated 1.5 g. of a brown powder containing 17,200 u/mg.

EXAMPLE? Five hundred forty-two mg. of the antibiotic ob- .tained in Example 6, containing 36,000 u/mg. are dis- .solved in 20 cc. of water, to give a solution of pH 2.3.

The pH is then adjusted to 6.85 by adding 8.9 cc. of 0.1N-sodium hydroxide solution, and the neutral solution is freeze-dried, to yield 565 mg. of the sodium salt of the antibiotic, whose aqueous solution displays a maximum ultraviolet absorption at A 256.5 mp. (E,,.,,,"' 106). Its elementary composition is as follows: C 47.4%, H 7.07%, O 35.3% (by difference),N=5.0l%, P= 1.84%,Na=3.4%.

14 EXAMPLE 8 Fifty g. of the antibiotic obtained in Example 5 are dissolved in 500 cc. of distilled water, and the solution is dialysed overnight against 40 liters of distilled water through a regenerated cellulose diaphragm.

After the dialysis, the solution of the antibiotic is mixed with 1 volume of n-propanol and the mixture is introduced into the top of a column charged, in order from the bottom, with g. of granulated carbon Acticarbone BS 40-80" previously washed with dilute hydrochloric acid, cc. of Amberlite [R 120 (H forrn), 500 g. of granulated carbon of the same quality as above.

When all of the antibiotic solution has percolated, elution is performed with n-propanoH-water (50:50 by volume). The active fractions (7 liters) are combined, neutralized to pH 7 with N-sodium hydroxide solution and concentrated to cc. under reduced pressure at a temperature below 40C. The concentrate is dialyzed for 24 hours against 4 X 10 liters of distilled water, and 1 volume of n-propanol is then added. The antibiotic is precipitated by adding 10 volumes of acetone. After isolation and drying, there are obtained 23.5 g. of the antibiotic in the form of its Na-salt containing 42,400 u/mg.

EXAMPLE 9 Twenty-two g. of the antibiotic obtained as described in Example 8 are dissolved in 110 cc. of water. The solution is mixed with 110 cc. of n-propanol and the mixture percolated through a column containing 100 cc. of alumina previously washed at pH 4. The development and the elution are perfonned with npropanol+water (50:50 by volume).

The active fractions are collected (430 cc.) and concentrated to 100 cc. under reduced pressure at a temperature below 40C. The concentrate is dialyzed overnight against 10 liters of distilled water. The dialysate is mixed with 1 volume of n-propanol and the active principle precipitated with 10 volumes of acetone. In this manner there are obtained 12 g. of purified antibiotic in the form of its sodium salt, containing 44,000 u/mg. and displaying the following characteristics:

C 45.0%, H 7.4%, O 37.6% (by difi'erence), N 3.72%,P=2.16%,Na=4.l5%.

Optical rotation [ab +6- '-1 (c 0.6 water) Ultraviolet spectrum: end absorption 220 m;.1.(E 30) 257 my. (E,* 3.5)

The present invention includes within its scope pharmaceutical compositions containing 11,837 RP. as such or as a non-toxic salt thereof, in conjunction with a compatible pharmaceutically acceptable carrier, which may itself by physiologically active. Such compositions may be in any pharmaceutical form suitable for the route of administration envisaged, and will ordinarily contain 5 to 95 percent by weight of the antibiotic compound.

Preparations according to the invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or suspending media are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. These compositions may also contain adjuvants such as preserving, wetting, emulsifying and dispersing agents. They may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporation in the compositions of sterilizing agents, by irradiation, or by heating.

The antibiotic may also be put up for clinical use as sterile solid, which can be dissolved in sterile water or some other sterile injectable medium immediately before use.

The proportion of active principle contained in the above-mentioned preparations may vary to suit the desired therapeutic effect. For treatment of infections by Gram-positive bacteria by intramuscular or intravenous administration, the dose varies in general from 0.25 to 1.5 g. for adults. This dose may be repeated either days later if the need should arise.

The following Example illustrates a pharmaceutical composition in accordance with the invention.

EXAMPLE 10 A solution is prepared which contains 50 g. of the sodium salt of 11,837 R.P. in distilled water to make 500 cc. This solution is sterilized by filtration through a bacteriostatic filter and then charged into ampoules (5 cc. per ampoule). The solution in the ampoules is then freeze-dried and the ampoules are sealed. For parenteral administration, the contents of the ampoules are dissolved immediately before use in 5 cc. of distilled water to make an injectable solution. In this manner, there are obtained about 5 cc. of solution containing 0.5 g. of the antibiotic.

WE CLAIM:

1. The antibiotic 1 1,837 RP. which is an acid of pKa 4.1 having a neutralization equivalent of about 600, very readily soluble in water, soluble in methanol, pyridine, acetic acid and dimethylforrnamide, and sparingly soluble to insoluble in ethanol, acetone, chloroform, and n-hexane, which decomposes without melting above 160C. and has the approximate elementary analysis C 46.9%, H 7.9%, O 39.2%, N 3.9% and P 2.25%, which in its infra-red spectrum, measured on a tablet of a mixture with KBr, shows the following maxima: 3360 very strong, 2930 strong, 1720 strong, 1630 very strong, 1564 medium, 1550 strong, 1430 shoulder, 1400 shoulder, 1380 strong, 1330 medium, 1222 strong, 1 164 medium, 1 125 shoulder, 1 100 medium, 1068 very strong, 1040 very su'ong, 968 medium, 950 medium, 890 weak, 860 weak and 800 weak, or the salts of the said antibiotic with bases.

2. An alkali metal salt of the antibiotic 1 1,837 R.P. of claim 1.

3. The sodium salt of 1 1,837 R.P. which has an optical rotation of [01],, +6 i 1, c 0.6, water, which has the elementary analysis C 45.0%, P1 7.4%, O 37.6%, N 3.72%, P 2.16% and Na 4.15%, which in its ultra-violet spectrum, measured on a 0.003 percent solution in water, shows end absorption at 220 mp. (E 30) and 257 my. (E, 3.5), and which in its infra-red spectrum, measured on a tablet of a mixture with KBr, shows the following maxima: 3400 very strong, 2910 strong, 1730 very strong, 1547 medium, 1525 strong, about 1430 shoulder, 1378 strong, 1330 medium, 1238 medium, 1160 medium, 1100 medium, 1062 very strong, 1042 shoulder, 1030 shoulder, 972 medium, 947 medium, 890 weak, and 860 weak.

4. Process for the production of the antibiotic 1 1,837 R.P. as defined in claim 1 which comprises aerobically cultivating Streptomyces viridans DS 9466 (NRRL 3087) on an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances, and separating the 1 1,837 R.P. formed during the cultivation.

5. Process according to claim 4, wherein the culture is efi'ected under submerged aerobic culture conditions at a pH between 6.0 and 7.8 and at a temperature from 23 to 35C. with an aeration rate of 0.3 to 2 liters of air per liter of medium per minute for from 4 to 7 days.

6. Process according to claim 5, wherein the pH of the nutrient medium at the beginning of the culture is between 6.5 and 7.5.

7. Process according to claim 5, wherein the temperature of the culture is 25 to 28C.

8. Process according to claim 5, wherein the l 1,8 37 RF. is separated from the culture medium by adjusting the pH of the medium to below 5, filtering and extracting the filter cake with water containing a lower alcohol atapllof3to 7.

9. Process according to claim 5, wherein the 11,837 R.P. is separated from the culture medium by adjusting the pH of the medium to below 5, filtering and extracting the filter cake with a mixture of lower alcohols.

10. Process according to claim 5, wherein the 1 1,837 R.P. is separated from the culture medium by adjusting the pH of the medium to about 7 and absorbing the antibiotic on to a strongly basic anion exchange resin from which it is eluted with an aqueous alcoholic solution containing an electrolyte.

11. Process according to claim 8, wherein the crude antibiotic is purified by chromatography on a strongly basic anion exchange resin.

12. Process according to claim 9, wherein the crude antibiotic is purified by chromatography on a strongly basic anion exchange resin.

13. Process according to claim 10, wherein the crude antibiotic is purified by chromatography on a strongly basic anion exchange resin.

14. Process according to claim 11, wherein the partially purified antibiotic obtained is further purified by dialysis of an aqueous solution thereof.

15. Process according to claim 12, wherein the plurality purified antibiotic obtained is further purified by dialysis of an aqueous solution thereof.

16. Process according to claim 13, wherein the partially purified antibiotic obtained is further purified by dialysis of an aqueous solution thereof.

17. A phannaceutical composition comprising, in association with a compatible pharmaceutically acceptable carrier, 5 to percent by weight of an antibiotic compound selected from the class consisting of the antibiotic 1 1,837 RP. which is an acid of pKa 4.1 having a neutralization equivalent of about 600, very readily soluble in water, soluble in methanol, pyridine, acetic acid and dimethylformamide, and sparingly soluble to insoluble in ethanol, acetone, chloroform, and nhexane, which decomposes without melting above C. and has the approximate elementary analysis C 46.9 percent, 11 7.9 percent, 0 39.2 percent, N 3.9 percent and P 2.25 percent, which in its infra-red spectrum, measured on a tablet of a mixture with KBr, shows the following maxima: 3360 very strong, 2930 weak and 800 weak, and the salts of the said antibiotic with non-toxic bases.

18. A pharmaceutical composition as claimed in claim 17 in the form of a sterile injectable solution. 

2. An alkali metal salt of the antibiotic 11,837 R.P. of claim
 3. The sodium salt of 11,837 R.P. which has an optical rotation of ( Alpha )D22 +6* + or - 1*, c 0.6, water, which has the elementary analysis C 45.0%, H 7.4%, O 37.6%, N 3.72%, P 2.16% and Na 4.15%, which in its ultra-violet spectrum, measured on a 0.003 percent solution in water, shows end absorption at 220 m Mu (E1 cm1% 30) and 257 m Mu (E1 cm1% 3.5), and which in its infra-red spectrum, measured on a tablet of a mixture with KBr, shows the following maxima: 3400 very strong, 2910 strong, 1730 very strong, 1547 medium, 1525 strong, about 1430 shoulder, 1378 strong, 1330 medium, 1238 medium, 1160 medium, 1100 medium, 1062 very strong, 1042 shoulder, 1030 shoulder, 972 medium, 947 medium, 890 weak, and 860 weak.
 4. Process for the production of the antibiotic 11,837 R.P. as defined in claim 1 which comprises aerobically cultivating Streptomyces viridans DS 9466 (NRRL 3087) on an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances, and separating the 11,837 R.P. formed during the cultivation.
 5. Process according to claim 4, wherein the culture is effected under submerged aerobic culture conditions at a pH between 6.0 and 7.8 and at a temperature from 23* to 35*C. with an aeration rate of 0.3 to 2 liters of air per liter of medium per minute for from 4 to 7 days.
 6. Process according to claim 5, wherein the pH of the nutrient medium at the beginning of the culture is between 6.5 and 7.5.
 7. Process according to claim 5, wherein the temperature of the culture is 25* to 28*C.
 8. Process according to claim 5, wherein the 11,837 R.P. is separated from the culture medium by adjusting the pH of the medium to below 5, filtering and extracting the filter cake with water containing a lower alcohol at a pH of 3 to
 7. 9. Process according to claim 5, wherein the 11,837 R.P. is separated from the culture medium by adjusting the pH of the medium to below 5, filtering and extracting the filter cake with a mixture of lower alcohols.
 10. Process according to claim 5, wherein the 11,837 R.P. is separated from the culture medium by adjusting the pH of the medium to about 7 and absorbing the antibiotic on to a strongly basic anion exchange resin from which it is eluted with an aqueous alcoholic solution containing an electrolyte.
 11. Process according to claim 8, wherein the crude antibiotic is purified by chromatography on a strongly basic anion exchange resin.
 12. Process according to claim 9, wherein the crude antibiotic is purified by chromatography on a strongly basic anion exchange resin.
 13. Process according to claim 10, wherein the crude antibiotic is purified by chromatography on a strongly basic anion exchange resin.
 14. Process according to claim 11, wherein the partially purified antibiotic obtained is further purified by dialysis of an aqueous solution thereof.
 15. Process according to claim 12, wherein the partially purified antibiotic obtained is further purified by dialysis of an aqueous solution thereof.
 16. Process according to claim 13, wherein the partially purified antibiotic obtained is further purified by dialysis of an aqueous solution thereof.
 17. A pharmaceutical composition comprising, in association with a compatible pharmaceutically acceptable carrier, 5 to 95 percent by weight of an antibiotic compound selected from the class consisting of the antibiotic 11,837 R.P. which is an acid of pKa 4.1 having a neutralization equivalent of about 600, very readily soluble in water, soluble in methanol, pyridine, acetic acid and dimethylformamide, and sparingly soluble to insoluble in ethanol, acetone, chloroform, and n-hexane, which decomposes without melting above 160*C. and has the approximate elementary analysis C 46.9 percent, H 7.9 percent, O 39.2 percent, N 3.9 percent and P 2.25 percent, which in its infra-red spectrum, measured on a tablet of a mixture with KBr, shows the following maxima: 3360 very strong, 2930 strong, 1720 strong, 1630 very strong, 1564 medium, 1550 strong, 1430 shoulder, 1400 shoulder, 1380 strong, 1330 medium, 1222 strong, 1164 medium, 1125 shoulder, 1100 medium, 1068 very strong, 1040 very strong, 968 medium, 950 medium, 890 weak, 860 weak and 800 weak, and the salts of the said antibiotic with non-toxic bases.
 18. A pharmaceutical composition as claimed in claim 17 in the form of a sterile injectable solution. 